BR112013009552B1 - RECOMBINANT MICROORGANISM PRODUCING 1-BUTANOL AND METHOD FOR PRODUCTION OF 1-BUTANOL - Google Patents

Abstract

PRODUCTION OF BUTANOL FROM CARBON MONOXIDE BY A RECOMBINANT MICRO-ORGANISM. The invention relates, in general, to new genetically modified microorganisms capable of using CO to produce 1-butanol and/or a precursor thereof, methyltransferases and new nucleic acids encoding the same, methods of producing genetically modified microorganisms using said new methyltransferases and methods of producing 1-butanol and/or a precursor thereof by means of microbial fermentation.

Description

FIELD

The present invention relates to methods for the production of biofuels by means of microbial fermentation and genetically modified microorganisms suitable for use in such methods.

BACKGROUND

Butanol is an important chemical with a wide range of industrial uses with a worldwide production of 4.5–5.5 million tonnes per year. It is used as a precursor for the production of acrylate and methacrylate esters (used in coatings, plastics, textiles, adhesives, etc.), glycol ethers (coatings, electronic components) and butyl acetate (paints, coatings, synthetic fruit flavours), as well as butylamines (production of pesticides and pharmaceuticals) and amine resins. It also has direct uses as a solvent (in paint, dyes, etc.), an extraction agent (for the production of pharmaceuticals and natural substances such as alkaloids, antibiotics, hormones and vitamins) and in de-icing fluids, cosmetics and chromatography.

Butanol also has potential as a second-generation biofuel and in this context is referred to as Biobutanol (Kopke & Dürre, 2010). It has properties similar to gasoline and superior to ethanol. Specifically, it has a high octane rating due to higher energy density, can be blended with gasoline at any concentration (whereas ethanol can be blended only up to 85%), and is non-hygroscopic and non-corrosive.

Biofuels for transportation are attractive substitutes for gasoline and are rapidly entering the market as low-concentration blends. Biofuels, derived from natural plant sources, are more environmentally sustainable than those derived from fossil resources (such as gasoline), their use allowing a reduction in the levels of so-called fossil carbon dioxide (CO2) gas that is released into the atmosphere as a result of fuel combustion. In addition, biofuels can be produced locally in many locations and can act to reduce dependence on imported fossil energy resources.

The vast majority of biofuels are produced via traditional yeast-based fermentation processes that use crop-derived carbohydrates as the primary carbon source and are known as first-generation biofuels. However, these crops are required for feed and many crops also require high agricultural inputs in the form of fertilizers. These limitations mean that first-generation biofuels are not considered sustainable and the greenhouse gas reductions that can be achieved are limited. The goal of secondary biofuels is the sustainable use of non-feed parts of current crops or other industrial wastes to reduce greenhouse gas emissions and reduce dependence on fossil fuels.

Recent production of 1-butanol has been mainly by oxo synthesis (Weiβermel & Arpe, 2003). Petrochemicals, including crude oil, are cracked to form propylene, which is used during oxo synthesis. However, the synthesis process requires the use of non-renewable natural resources, as well as being expensive and non-specific in the products formed.

Butanol can also be produced through biological production methods, the most common being Acetone-Butanol-Ethanol (ABE) fermentation, which has been used industrially since 1913 (Kõpke & Dürre, 2010). This method has the undesirable byproduct of acetone, which is usually produced at about half the volume of butanol, thus substantially reducing the yield. Additionally, this fermentation method is limited by the toxicity of butanol to the microorganism, resulting in growth being almost completely inhibited at butanol concentrations as low as 1.5% (Kõpke and Dürre 2010). Furthermore, ABE fermentation uses corn sugar, starch, cassava, and sugarcane as feedstocks. This results in the undesirable use of arable land to produce fuel rather than food. This can also aggravate problems related to deforestation and desertification.

Only a few organisms are known to naturally produce butanol, and none of these produce butanol in high yields from abundant sources (such as carbon monoxide - CO). Two organisms known to naturally produce butanol from CO are Butyribacterium methylotrophicum (which synthesizes only trace amounts of butanol (Heiskanen et al., 2007)) and Clostridium carboxidivorans (which produces low yields of 1-butanol as a byproduct to the major fermentation products of ethanol and acetate (Liou et al., 2005)).

A number of organisms have been genetically engineered to produce 1-butanol, including E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Pseudomonas putida or Lactobacillus brevis. However, all of these organisms still rely on sugar as a raw material (Kõpke & Dürre, 2010). Although over 250 species of Clostridium are known, only a few are genetically accessible. There is no known natural competence (uptake of extracellular DNA from the cell environment) in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues pose significant difficulties in the effective transformation of Clostridium species. Most Clostridia have one or more restriction/methylation systems to protect against foreign and phage DNA, which means that transformation is particularly difficult and unpredictable.

Bibliographic details of the publications mentioned here are compiled at the end of the description.

It is an object of the invention to overcome one or more drawbacks of the prior art or at least to provide the public with a useful alternative to known technologies.

SUMMARY OF THE INVENTION

According to the invention, it has been discovered that a genetically modified microorganism is capable of using CO to produce 1-butanol or a precursor thereof as the main fermentation product.

In a first aspect, the invention provides a recombinant acetogenic carboxytrophic microorganism that produces 1-butanol and/or a precursor thereof as the main fermentation product.

In a related aspect, the invention provides a recombinant acetogenic microorganism that is capable of producing 1-butanol and/or a precursor thereof by fermentation of a substrate comprising CO at a concentration greater than approximately 1 mM or 0.075 g/L per liter of fermentation broth.

Preferably, the microorganism comprises exogenous nucleic acids adapted to express one or more enzymes in the butanol biosynthesis pathway.

In one embodiment, the one or more enzymes are selected from the group consisting of: Thiolase 3-hydroxybutyryl-CoA dehydrogenase Crotonyl-CoA crotonase/hydratase Butyryl-CoA dehydrogenase Electron Transfer Flavoprotein A Electron Transfer Flavoprotein B

Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes.

Preferably, the one or more nucleic acids encoding one or more enzymes are chosen from nucleic acids SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.

Preferably, the microorganism comprises one or more exogenous nucleic acids encoding each of thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, and Electron Transfer Flavoprotein B.

Preferably, the microorganism comprises a plasmid encoding one or more of, or preferably each of, Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.

In one embodiment, the microorganism comprises one or more exogenous nucleic acids encoding each of the enzymes thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase, and butyryl-CoA dehydrogenase.

Preferably, the microorganism further comprises an exogenous acetate kinase/phosphotransacetylase promoter. Preferably, the promoter corresponds to SEQ ID NO. 7 or a functionally equivalent variant thereof.

Preferably, the promoter is contained in a construct encoding one or more of the enzymes mentioned above.

In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of the enzymes selected from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin-dependent aldehyde oxidoreductase; butyraldehyde dehydrogenase; butanol dehydrogenase; a butyraldehyde dehydrogenase and bifunctional butanol dehydrogenase.

In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.

In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase. In particular embodiments, the microorganism comprises exogenous nucleic acids adapted to express each of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase.

In one embodiment, the one or more nucleic acids encoding one or more enzymes are chosen from the nucleic acids described in Tables 7 to 10 hereinafter and functionally equivalent variants thereof.

In one embodiment, the microorganism comprises one or more nucleic acids suitable for expressing at least two of the enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.

In one embodiment, the microorganism comprises one or more nucleic acids suitable for expressing thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase/crotonyl-CoA hydratase, butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, Electron Transfer Flavoprotein B, and one or both of butyraldehyde dehydrogenase and butanol dehydrogenase (or a bifunctional enzyme).

In one embodiment, the microorganism comprises one or more nucleic acids suitable for expressing thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase/crotonyl-CoA hydratase, butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, Electron Transfer Flavoprotein B, and at least one of phosphotransbutyrylase and butyrate kinase and ferredoxin-dependent aldehyde oxidoreductase and butanol dehydrogenase.

Preferably, the microorganism is selected from the group of carboxytrophic acetogenic bacteria. In certain embodiments, the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium Ijungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanae- robacter kiuvi. Preferably, the microorganism is Clostridium autoethanogeneum DSM23693.

In one embodiment, the recombinant microorganism of the invention has the characteristics that define the microorganism deposited in the DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) under accession number DSM24138.

In a second aspect, the invention provides a recombinant methyltransferase gene according to Nucleotide SEQ ID NO. 27 or a functionally equivalent variant thereof.

In a third aspect, the invention provides a methyltransferase according to SEQ ID NO. 28 or a functionally equivalent amino acid variant thereof.

In a related aspect, the invention provides a recombinant microorganism comprising a methyltransferase gene according to the second aspect. The methyltransferase gene may be present in a nucleic acid construct or integrated into the genome of the microorganism.

In a fourth aspect, the invention provides a nucleic acid comprising SEQ ID NO. 1 to 6 or functionally equivalent variants thereof, in any order.

Preferably, the nucleic acid comprises SEQ ID NO. 1 to 6, in the order shown in Figure 2.

Preferably, the nucleic acid further comprises a butyryl/acetyl phosphate transferase promoter. Preferably, the promoter corresponds to SEQ ID NO. 7 or a functionally equivalent variant thereof.

In a fifth aspect, the invention provides an expression construct comprising one or more nucleic acid sequences wherein the construct, when expressed in an acetogenic microorganism, results in 1-butanol and/or a precursor thereof being produced as the major fermentation product.

Preferably, the one or more nucleic acid sequences encode one or more enzymes that are part of the 1-butanol biosynthesis pathway.

Preferably, the nucleic acids are selected from nucleic acids encoding thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, electron transfer Flavoprotein A and/or electron transfer Flavoprotein B.

Preferably, the one or more nucleic acid sequences are selected from SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.

In one embodiment, the nucleic acids are further selected from nucleic acids encoding phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.

In one embodiment, the nucleic acids are selected from the group of nucleic acids described in Tables 7 to 10 hereinafter and functionally equivalent variants thereof.

In one embodiment, the expression construct encodes at least two enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.

Preferably, the expression construct further comprises a butyryl/acetyl phosphate transferase operon promoter. In another embodiment, the expression construct comprises another highly active promoter, such as the ferredoxin:pyruvate oxidoreductase promoter (SEQ ID NO. 48), the Wood-Ljungdahl gene cluster (SEQ ID NO. 47), the Rnf operon (SEQ ID NO. 49), or the ATP synthase operon (SEQ ID NO. 50). Preferably, the butyryl/acetyl phosphate transferase operon promoter corresponds to SEQ ID NO. 7 or a functionally equivalent variant thereof.

In a sixth aspect, the invention provides a construct comprising a methyltransferase gene as described herein.

In a seventh aspect, the invention provides a composition comprising the expression construct of the fifth aspect and the methylation construct of the sixth aspect.

Preferably, the composition is capable of producing a recombinant microorganism that produces 1-butanol and/or a precursor thereof as the main fermentation product.

In an eighth aspect, the invention provides a method of producing a recombinant microorganism comprising: a. introducing into a transport microorganism (i) an expression construct and (ii) a methylation construct according to the sixth aspect comprising a methyltransferase gene; b. expressing the methyltransferase gene; c. isolating one or more constructs from the transport microorganism; and d. introducing at least one expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.

In one embodiment, the methylation construct and the expression construct are both isolated in step c. In another embodiment, the expression construct is isolated in step c.

In one embodiment, only the expression construct is introduced into the target microorganism. In another embodiment, the expression construct and the methylation construct are both introduced into the target microorganism.

Preferably, the expression construct is as defined in the fifth aspect.

Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.

In a related aspect, the invention provides a method of producing a recombinant microorganism comprising: a. methylating an expression construct in vitro by a methyltransferase according to SEQ ID NO. 28 or a functionally equivalent variant thereof; b. introducing an expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism. Preferably, the expression construct is as defined in the fifth aspect.

Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.

Preferably, the methyltransferase is produced by expressing a methyltransferase gene, preferably according to SEQ ID NO. 27 or a functionally equivalent variant thereof, in a microorganism and isolating the methyltransferase enzyme.

In another related aspect, the invention provides a method of producing a recombinant microorganism comprising: a. introducing into the genome of a transport microorganism a methyltransferase gene, preferably according to SEQ ID NO. 27 or a functionally equivalent variant thereof; b. introducing an expression construct into the transport microorganism; c. isolating one or more constructs from the transport microorganism; and d. introducing at least one expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

Preferably, the expression construct is as defined in the fifth aspect.

Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.

In another related aspect, the invention provides a method of producing a recombinant microorganism comprising: a. methylating an expression construct according to the fifth aspect in vitro by a methyltransferase; b. introducing the expression construct into a target microorganism.

Preferably, the methyltransferase is encoded by a methyltransferase gene as defined in the second aspect or a methyltransferase as defined in the third aspect.

Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.

In a ninth aspect, the invention provides a method of producing a recombinant microorganism comprising: a. introducing (i) an expression construct according to the fifth aspect and (ii) a methylation construct comprising a methyltransferase gene into a transport microorganism; b. expressing the methyltransferase gene; c. isolating one or more constructs from the transport microorganism; and d. introducing at least one expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.

In one embodiment, the methylation construct and the expression construct are both isolated in step c. In another embodiment, the expression construct is isolated in step c.

In one embodiment, only the expression construct is introduced into the target microorganism. In another embodiment, the expression construct and the methylation construct are both introduced into the target microorganism.

Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.

In a tenth aspect, the invention provides a method of producing a recombinant microorganism that produces 1-butanol or a precursor thereof as the main fermentation product comprising: a. introducing (i) an expression construct and (ii) a methylation construct comprising a methyltransferase gene into a transport microorganism; b. expressing the methyltransferase gene; c. isolating one or more constructs from the transport microorganism; and d. introducing at least one expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.

In one embodiment, the methylation construct and the expression construct are both isolated in step c. In another embodiment, the expression construct is isolated in step c.

In one embodiment, only the expression construct is introduced into the target microorganism. In another embodiment, the expression construct and the methylation construct are both introduced into the target microorganism.

Preferably, the expression construct is as defined in the fifth aspect.

Preferably, the methylation construct is as defined in the sixth aspect.

In an eleventh aspect, the invention provides a method of producing 1-butanol and/or a precursor thereof by microbial fermentation comprising fermentation of a substrate using a recombinant microorganism.

Preferably, 1-butanol and/or a precursor thereof is the main fermentation product.

Preferably, the recombinant microorganism is as described in any one of the eighth to tenth aspects.

Preferably, 1-butanol and/or a precursor thereof is produced in a yield of approximately 0.075 grams per liter of fermentation broth (g/L) to about 20 g/L. In one embodiment, the yield is approximately 0.15 g/L to about 1.54 g/L. In other embodiments, the yield is approximately 10 g/L, approximately 5 g/L, or approximately 2 g/L. Preferably, the yield of 1-butanol is up to the limit at which the butanol becomes toxic to the surrounding medium.

Preferably, the substrate comprises CO. Preferably, the substrate is a gaseous substrate comprising CO. In one embodiment, the substrate comprises an industrial waste gas. In certain embodiments, the gas is waste gas or syngas from steel milling.

In one embodiment, the substrate will typically contain a large proportion of CO, such as at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.

While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to the formation of product according to the methods of the invention. In particular embodiments, the presence of hydrogen results in an overall increased efficiency of alcohol production. For example, in particular embodiments, the substrate can comprise an approximate H2/CO ratio of 2:1 or 1:1 or 1:2. In one embodiment, the substrate comprises about 30% or less H2 by volume, 20% or less H2 by volume, about 15% or less H2 by volume, or about 10% or less H2 by volume. In other embodiments, the substrate stream comprises low concentrations of H2, e.g., less than 5% or less than 4% or less than 3% or less than 2% or less than 1%, or is substantially free of hydrogen. The substrate may also contain some CO2, for example, such as about 1% to about 80% CO2 by volume or 1% to about 30% CO2 by volume.

Preferably, the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase.

Preferably, the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase, both before and after introduction of an exogenous nucleic acid.

Preferably, the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.

Preferably, the microorganism produces butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase before and after introduction of an exogenous nucleic acid.

In a twelfth aspect, the invention provides 1-butanol or a precursor thereof produced by the method of the eleventh aspect.

In a thirteenth aspect, the invention provides a transport microorganism comprising a methylation construct as defined herein.

Preferably, the transport microorganism further comprises an expression construct as defined herein.

Preferably, the transport microorganism is E. coli, Bacillus subtillis or Lactococcus lactis.

Preferably, the methylation construct of any of the foregoing aspects comprises a lac promoter and the methyltransferase gene and is induced by isopropyl-β-thio-D-galactoside (IPTG). Methyltransferase expression may also be controlled by other inducible promoter systems, such as ara, tet or T7.

In a fourteenth aspect, the invention provides a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs. 8 to 13.

In a fifteenth aspect, the invention provides a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs. 16 to 23.

In a sixteenth aspect, the invention provides a nucleic acid comprising at least the nucleic acid sequence of SEQ ID NO. 7 or a functionally equivalent variant thereof, a nucleic acid construct or vector comprising the same, and microorganisms comprising said nucleic acid or nucleic acid construct or vector.

In a seventeenth aspect, the invention provides a nucleic acid which encodes a methyltransferase according to SEQ ID NO. 28.

In an eighteenth aspect, the invention provides a nucleic acid comprising a nucleic acid encoding a polypeptide having the amino acid sequence of a polypeptide chosen from the groups listed in Tables 7 to 10 hereinafter and functionally equivalent variants of any one or more thereof.

In a nineteenth aspect, the invention provides a nucleic acid comprising a nucleic acid chosen from the groups listed in Tables 7 to 10 hereinafter and functionally equivalent variants of any one or more thereof.

In a twentieth aspect, the invention provides constructs and microorganisms comprising a nucleic acid of the eighteenth or nineteenth aspects of the invention.

In a twenty-first aspect, the invention provides a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs. 32 to 38 and 123 to 135.

In a twenty-second aspect, the invention provides a polypeptide comprising the amino acid sequence of a polypeptide chosen from the groups listed in Tables 7 to 10 hereinafter and functionally equivalent variants of any one or more thereof.

The invention may also be referred to broadly as consisting of the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, or any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have equivalents known in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE FIGURES

These and other aspects of the present invention, which should be considered in all their novel aspects, will be apparent from the following description, which is given by way of example only, with reference to the accompanying Figures, in which:

Figure 1 shows the biosynthesis pathway of butanol from CO.

Figure 2 shows an expression plasmid encoding exemplary genes involved in the biosynthesis of 1-butanol.

Figure 3 shows the sequencing results of pM-TL85245-thlA-crt-hbd, which demonstrate that the 1-butanol biosynthesis genes found in the expression plasmid were free of mutations.

Figures 4a, 4b, and 4c show a nucleotide alignment of C. autoethanogenum (CAU), C. Ijungdahlii (CLJ), C. ragsdalei (CRA), and the designed methyltransferase (DMT) genes.

Figure 4d shows an amino acid alignment of the methyltransferases from C. autoethanogenum (CAU 1+2), C. Ijungdahlii (CLJ), C. ragsdalei (CRA1+2) and the designed methyltransferase (DMT).

Figure 5 shows an exemplary methylation plasmid of the invention.

Figure 6 shows an agarose gel electrophoresis image of isolated plasmid DNA. Rows 1, 6, 11, 16, 21, and 26 show 100 bp Ladder Plus DNA. Rows 2–5 show PCR with original methylated plasmid mixture as template in the following order: ermB, ColE1, thIA, crt. Rows 7–10, 12–15, 17–20, 22–25, and 27–30 show PCR with plasmids isolated from 4 different clones as template, each in the following order: ermB, ColE1, thIA, crt. Rows 32–35 show prep plasmid from 4 different clones. Row 36 shows prep plasmid from original C. autoethanogenum DSM23693.

Figure 7 shows HPLC results showing the production of 1-butanol with C. autoethanogenum harboring the butanol plasmid pMTL85245-thlA-CRT-hbd.

Figure 8 shows an expression analysis of over 200 genes during a typical fermentation with Clostridium autoethanogenum under conventional conditions using real-time PCR to identify promoter regions suitable for heterologous gene expression.

Figure 9 shows the sequence of SEQ ID NOs. 1, 2, and 3.

Figure 10 shows the sequence of SEQ ID NOs. 4, 5, and 6.

Figure 11 shows the sequence of promoter regions encoded by SEQ ID NOs. 7, 47, 48, 49, and 50.

Figure 12 shows the sequence of SEQ ID NO. 14.

Figure 13 shows the sequence of SEQ ID NO. 15.

Figure 14 shows the sequence of SEQ ID NOs. 24 and 25.

Figure 15 shows the sequence of SEQ ID NO. 26.

Figure 16 shows the sequence of SEQ ID NO. 27.

Figure 17 shows the sequence of SEQ ID NO. 28.

Figure 18 shows the sequence of SEQ ID NO. 29.

Figure 19 shows the 16s rRNA gene of C. autoethanogenum (Y18178, Gl: 7271109)

Figures 20 and 21 show the sequence of SEQ ID NO. 31.

Figure 22 shows Seq. ID 39: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. autoethanogenum.

Figure 23 shows Seq. ID 40: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. autoethanogenum.

Figure 24 shows Seq. ID 41: nucleotide sequence of butyraldehyde dehydrogenase from C. autoethanogenum and Seq. ID 42: amino acid sequence of butyraldehyde dehydrogenase from C. autoethanogenum.

Figure 25 shows Seq. ID 43: nucleotide sequence of butyraldehyde dehydrogenase from C. autoethanogenum and Seq. ID 44: amino acid sequence of butyraldehyde dehydrogenase from C. autoethanogenum.

Figure 26 shows Seq. ID 45: nucleotide sequence of butyraldehyde dehydrogenase from C. autoethanogenum.

Figure 27 shows Seq. ID 46: amino acid sequence of butyraldehyde dehydrogenase from C. autoethanogenum and Seq. ID 119: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 28 shows Seq. ID 120: amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 121: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 29 shows Seq. ID 122: amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 51: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 30 shows Seq. ID 52: amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 53: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 31 shows Seq. ID 54: Amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 55: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 32 shows Seq. ID 56: Amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 57: nucleotide sequence of butanol dehydrogenase from C. autoethanogenum.

Figure 33 shows Seq. ID 58: amino acid sequence of butanol dehydrogenase from C. autoethanogenum and Seq. ID 59: nucleotide sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum and Seq. ID 60: amino acid sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum.

Figure 34 shows Seq. ID 61: nucleotide sequence of C. autoethanogenum acetate/butyrate kinase and Seq. ID 62: amino acid sequence of C. autoethanogenum acetate/butyrate kinase.

Figure 35 shows Seq. ID 63: nucleotide sequence of aldehyde:ferredoxin oxide reductase from C. autoethanogenum and Seq. ID 64: amino acid sequence of aldehyde:ferredoxin oxide reductase from C. autoethanogenum.

Figure 36 shows Seq. ID 65: nucleotide sequence of aldehyde:ferredoxin oxide reductase from C. autoethanogenum and Seq. ID 66: amino acid sequence of aldehyde:ferredoxin oxide reductase from C. autoethanogenum.

Figure 37 shows Seq. ID 67: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. Ijungdahlii

Figure 38 shows Seq. ID 68: amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. Ijungdahlii

Figure 39 shows Seq. ID 69: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. Ijungdahlii

Figure 40 shows Seq. ID 70: amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. Ijungdahlii and Seq. ID 71: nucleotide sequence of butyraldehyde dehydrogenase from C. Ijungdahlii.

Figure 41 shows Seq. ID 72: amino acid sequence of butyraldehyde dehydrogenase from C. Ijungdahlii and Seq. ID 73: nucleotide sequence of butyraldehyde dehydrogenase from C. Ijungdahlii and Seq. ID 74: amino acid sequence of butyraldehyde dehydrogenase from C. Ijungdahlii.

Figure 42 shows Seq. ID 75: nucleotide sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 76: amino acid sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 77: nucleotide sequence of butanol dehydrogenase from C. Ijungdahlii.

Figure 43 shows Seq. ID 78: amino acid sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 79: nucleotide sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 80: amino acid sequence of butanol dehydrogenase from C. Ijungdahlii.

Figure 44 shows Seq. ID 81: nucleotide sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 82: amino acid sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 83: nucleotide sequence of butanol dehydrogenase from C. Ijungdahlii.

Figure 45 shows Seq. ID 84: amino acid sequence of butanol dehydrogenase from C. Ijungdahlii and Seq. ID 85: nucleotide sequence of phosphate acetyl/butyryl transferase from C. Ijungdahlii and Seq. ID 86: amino acid sequence of phosphate acetyl/butyryl transferase from C. Ijungdahlii and Seq. ID 87: nucleotide sequence of acetate/butyrate kinase from C. Ijungdahlii.

Figure 46 shows Seq. ID 88: amino acid sequence of acetate/butyrate kinase from C. Ijungdahlii and Seq. ID 89: nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. Ijungdahlii and Seq. ID 90: amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. Ijungdahlii.

Figure 47 shows Seq. ID 91: nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. Ijungdahlii and Seq. ID 92: amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. Ijungdahlii.

Figure 48 shows Seq. ID 93: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei

Figure 49 shows Seq. ID 94: amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei

Figure 50 shows Seq. ID 95: nucleotide sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei.

Figure 51 shows Seq. ID 96: amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei and Seq. ID 97: nucleotide sequence of butyraldehyde dehydrogenase from C. ragsdalei.

Figure 52 shows Seq. ID 98: amino acid sequence of C. ragsdalei butyraldehyde dehydrogenase, Seq. ID 99: nucleotide sequence of C. ragsdalei butyraldehyde dehydrogenase, and Seq. ID 100: amino acid sequence of C. ragsdalei butyraldehyde dehydrogenase.

Figure 53 shows Seq. ID 101: nucleotide sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 102: amino acid sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 103: nucleotide sequence of butanol dehydrogenase from C. ragsdalei.

Figure 54 shows Seq. ID 104: amino acid sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 105: nucleotide sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 106: amino acid sequence of butanol dehydrogenase from C. ragsdalei:

Figure 55 shows Seq. ID 107: nucleotide sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 108: amino acid sequence of butanol dehydrogenase from C. ragsdalei and Seq. ID 109: nucleotide sequence of butanol dehydrogenase from C. ragsdalei.

Figure 56 shows Seq. ID 110: amino acid sequence of C. ragsdalei butanol dehydrogenase and Seq. ID 111: nucleotide sequence of C. ragsdalei phosphate acetyl/butyryl transferase and Seq. ID 112: amino acid sequence of C. ragsdalei phosphate acetyl/butyryl transferase and Seq. ID 113: nucleotide sequence of C. ragsdalei acetate/butyrate kinase.

Figure 57 shows Seq. ID 114: amino acid sequence of C. ragsdalei acetate/butyrate kinase and Seq. ID 115: nucleotide sequence of C. ragsdalei aldehyde:ferredoxin oxidoreductase and Seq. ID 116: amino acid sequence of C. ragsdalei aldehyde:ferredoxin oxidoreductase.

Figure 58 shows Seq. ID 117: nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei and Seq. ID 118: amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei.

Figure 59 shows SEQ ID 136: the 16s rRNA gene from Clostridium Ijungdahlii (CP001666.1, Gl: 300433347).

Figure 60 shows the gene expression pattern of (A) de-

bifunctional butyraldehyde/butanol hydrogenase (Seq ID 39); (B) butyraldehyde dehydrogenase (SEQ ID 41); (C) butyraldehyde dehydrogenase (SEQ ID 45); (D) butanol dehydrogenase (SEQ ID 53); (E) butanol dehydrogenase (SEQ ID 57); (F) acetyl/butyryl phosphate transferase (SEQ ID 57); (G), acetate/butyrate kinase (SEQ ID 59); (H) aldehydeferredoxin oxidoreductase (SEQ ID 63); (I) aldehyde-ferredoxin oxidoreductase (SEQ ID 65).

DETAILED DESCRIPTION OF THE INVENTION

The following is a description of the present invention, including preferred embodiments thereof, provided in general terms. The invention is further elucidated from the description provided under the heading "Examples" herein below, which provide experimental data supporting the invention, specific examples of various aspects of the invention, and means of carrying out the invention.

Among others, the closely related microorganisms C. autoethanogenum, C. Ijungdahlii and C. ragsdalei are known to be useful for the production of ethanol as a biofuel from carbon monoxide. In order to produce 1-butanol as a biofuel from a gaseous substrate, a universal transformation system for these organisms was developed and production of 1-butanol as the major fermentation product from CO was demonstrated.

The inventors discovered that when particular genes encoding proteins in the 1-butanol biosynthetic pathway (Figure 1) were introduced into acetogenic microorganisms, these microorganisms were able to use a gaseous substrate to produce 1-butanol or a precursor thereof as the major fermentation product. Although some unmodified microorganisms are known to produce 1-butanol, the yield of 1-butanol from CO produced by these unmodified microorganisms is very low. As a result, its usefulness for the production of biofuels from gaseous substrates is extremely limited due to its low efficiency and subsequent lack of commercial viability. Clostridium autoethanogenum naturally produces ethanol, acetate, 2,3-butanediol, and lactic acid, but is not known to produce 1-butanol.

As shown in Figure 1, the Wood-Ljungdahl pathway converts CO to acetyl-CoA. This compound can be further converted to 1-butanol in acetogenic microorganisms by the action of the enzymes thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and butanol dehydrogenase. In a particular embodiment of the invention, the microorganism expresses the first four enzymes, which can be encoded by nucleic acids SEQ ID NOs. 1 to 4 or functionally equivalent variants thereof. The present invention provides a microorganism that facilitates the conversion of acetyl-CoA to 1-butanol by the action of enzymes encoded by recombinant nucleic acids as well as naturally occurring enzymes. The invention also provides the use of microorganisms expressing other recombinant nucleic acid sequences which encode enzymes at other stages in the butanol or Wodd-Ljungdahl biosynthesis pathways. The inventors have also identified a number of novel enzymes and nucleic acids.

Since there is no known natural competence (uptake of extracellular DNA from the cell environment) in Clostridia, electrotransformation or conjugation are the only methods available for transformation. These issues pose significant difficulties in the effective transformation of Clostridium species. Additionally, the restriction/methylation systems found in Clostridia protect against foreign and phage DNA and result in their genetic transformation being particularly problematic. It has been shown that transformation of several Clostridium strains (C. acetobutylicum ATCC824, C. cellulolyticum ATCC35319, C. botulinum ATCC25765 and C. difficile CD3 and CD6) is possible only if the DNA is methylated in vivo in E. coli or methylated in vitro in a specific pattern prior to transformation (Memelstein et al., 1993; Herbert et al., 2003; Jennert et al., 2000; Davis et al., 2000). However, determination of the correct methylation pattern is often not possible due to nonspecific exohucleases, etc. Additionally, many Clostridium species also possess restriction systems which digest DNA that is methylated at a specific (“wrong”) position.

The above-mentioned major obstacles have been overcome by the inventors in developing the recombinant microorganisms of the present invention. A novel methylation system comprising a novel methytransferase gene has been developed to circumvent the naturally occurring restriction barriers present in native acetogenic microorganisms. Accordingly, the methylation method and methyltransferase gene of the present invention can be applied to a variety of compatible microorganisms that possess barriers that prevent the effective introduction and expression of desirable recombinant nucleic acids into microorganisms.

Definitions

As noted herein, "1-butanol precursors" include butyryl-CoA, butyryl-phosphate, butyrate, and butyraldehyde.

As referred to herein, a "fermentation broth" is a culture medium comprising at least nutrient media and bacterial cells.

As referred to here, a "transport microorganism" is a microorganism in which a methyltransferase enzyme is expressed and is distinct from the target microorganism.

As referred to herein, a "target microorganism" is a microorganism in which the genes included in the expression construct are expressed and is distinct from the transport microorganism.

As referred to herein, the term "principal fermentation product" is intended to mean the first fermentation product which is produced in greatest concentration and/or yield.

The terms "increased efficiency", "increased efficiency" and the like, when used in relation to a fermentation process include, but are not limited to, increasing one or more of the growth rate of microorganisms that catalyze the fermentation, the volume of desired product (such as alcohols) produced per volume of substrate (such as sugar) consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared to other by-products of the fermentation.

The phrase "substrate comprising carbon monoxide" and similar terms should be understood to include any substrate on which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.

The phrase "gaseous substrate comprising carbon monoxide" and similar phrases and terms include any gas which contains a level of carbon monoxide. In certain embodiments, the substrate contains at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.

While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to the formation of the product according to the methods of the invention. In particular embodiments, the presence of hydrogen results in an overall increased efficiency of alcohol production. For example, in particular embodiments, the substrate may comprise an approximate H2:CO ratio of 2:1 or 1:1 or 1:2. In one embodiment, the substrate comprises about 30% or less H2 by volume, 20% or less H2 by volume, about 15% or less H2 by volume, or about 10% or less H2 by volume. In other embodiments, the substrate stream comprises low concentrations of H2, e.g., less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially free of hydrogen. The substrate may also contain some CO2, for example, such as about 1% to about 80% CO2 by volume or 1% to about 30% CO2 by volume. In one embodiment, the substrate comprises less than or equal to about 20% CO2 by volume. In particular embodiments, the substrate comprises less than or equal to about 15% CO2 by volume, less than or equal to about 10% CO2 by volume, less than or equal to about 5% CO2 by volume, or substantially no CO2.

In the description which follows, embodiments of the invention are described in terms of the delivery and fermentation of a "CO-containing gaseous substrate". However, it should be noted that the gaseous substrate may be supplied in alternative forms. For example, the CO-containing gaseous substrate may be supplied dissolved in a liquid. Essentially, a liquid is saturated with a gas containing carbon monoxide and then this liquid is added to the bioreactor. This may be accomplished using standard methodology. By way of example, a microbubble dispersion generator (Hensirisak et al., Scale-Up of Microbubble Dispersion Generator For Aerobic Fermentation; Applied Biochemistry and Biotechnology Volume 101, Issue 3 / October 2002) could be used. By way of further example, the CO-containing gaseous substrate may be adsorbed onto a solid support. Such alternative methods are encompassed by the use of the term "CO-containing substrate" and the like.

In particular embodiments of the invention, the CO-containing gaseous substrate is a waste or industrial gas or effluent. "Waste or industrial gas or effluent" should be taken broadly to include any gas comprising CO produced by an industrial process and includes gases produced as a result of ferrous metal product manufacturing, non-ferrous product manufacturing, petroleum refining processes, coal gasification, biomass gasification, electrical power production, carbon black production, and coke manufacturing. Other examples may be provided elsewhere herein.

Unless the context otherwise requires, the phrases "fermentation", "fermentation process" or "fermentation reaction" and the like, as used herein, are intended to encompass both the growth phase and the product biosynthesis phase of the process. As will be described in greater detail below, in some embodiments, the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, the addition of metals or compositions to a fermentation reaction should be understood to include addition to one or both of these reactors.

The term "bioreactor" includes a fermentation device consisting of one or more vessels and/or towers or piping configuration, which includes a Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer or other vessel or other device suitable for gas-liquid contact. As further described herein, in some embodiments, the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, when referring to the addition of substrate to the bioreactor or fermentation reaction, this should be understood to include addition to one or both of these reactors, where appropriate. "Exogenous nucleic acids" are nucleic acids which originate outside the microorganism into which they are to be introduced. Exogenous nucleic acids may be derived from any suitable source including, but not limited to, the microorganism into which they are to be introduced, strains or species of microorganisms that differ from the organism into which they are to be introduced, or they may be artificially or recombinantly created. In one embodiment, exogenous nucleic acids represent nucleic acid sequences naturally present in the microorganism into which they are to be introduced and they are introduced to enhance the expression or overexpression of a particular gene (e.g., by increasing the copy number of the sequence (e.g., a gene)). In another embodiment, the exogenous nucleic acids represent nucleic acid sequences not naturally present within the microorganism into which they are to be introduced and allow for the expression of a product not naturally present in the microorganism or increased expression of a gene native to the microorganism (e.g., in the case of introduction of a regulatory element, such as a promoter). The exogenous nucleic acid may be adapted to integrate into the genome of the microorganism into which it is to be introduced or remain in an extrachromosomal state.

It will be appreciated that the invention may be practiced using nucleic acids whose sequence differs from the sequences specifically exemplified herein, so long as they perform substantially the same function. For nucleic acid sequences encoding a protein or peptide, this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences representing promoter sequences, the variant sequence will have the ability to promote the expression of one or more genes. Such nucleic acids may be referred to herein as "functionally equivalent variants." By way of example, functionally equivalent variants of a nucleic acid include allelic variants, fragments of a gene, genes which include mutations (deletion, insertion, nucleotide substitutions, and the like) and/or polymorphisms, and the like. Homologous genes from other bacteria capable of butyric acid or butanol fermentation may also be considered as examples of functionally equivalent variants of the sequences specifically exemplified herein. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium tetani, Clostridium pasteurianum, Clostridium kluyveri, Clostridium cellulovorans, Clostridium perfringens, Clostridium botulinum, Clostridium butyricum strain DSM10702, Clostridium tyrobutyricum strain ATCC 25755, Anaerococcus prevotii DSM 20548, Thermoanaerobacter tengcongensis, Brachyspira pilosicoli, Bacillus megaterium, Streptococcus pyogenes and Clostridium saccharoperbutylacetonicum, details of which are publicly available on websites such as Genbank or NCBI. The phrase "functionally equivalent variants" should also be taken to include nucleic acids whose sequence varies as a result of codon optimization for a particular organism. "Functionally equivalent variants" of a nucleic acid herein will preferably have at least about 70%, preferably about 80%, more preferably about 85%, preferably about 90%, most preferably about 95% or more nucleic acid sequence identity to the identified nucleic acid. In a particular embodiment, the functionally equivalent variant of the thiolase gene as defined herein may be the atoAB gene in E. coli (NC-000913.2; atoA = GenelD: 946719; atoB = GenelD: 946727). Functionally equivalent variants of the eftAB gene as defined herein can be found in Tsai and Saier (1995).

It will also be appreciated that the invention may be practiced using polypeptides whose sequence differs from the amino acid sequences specifically exemplified herein. Such variants may be referred to herein as "functionally equivalent variants." A functionally equivalent variant of a protein or a peptide includes those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or more amino acid identity with the identified protein or peptide and have substantially the same function as the peptide or protein of interest. Such variants include, within their scope, fragments of a protein or a peptide, wherein the fragment comprises a truncated form of the polypeptide, wherein deletions may be from 1 to 5, to 10, to 15, to 20, to 25 amino acids and may extend from residue 1 to 25 at either terminus of the polypeptide and wherein deletions may be of any length within the region; or may be in an internal location. Functionally equivalent variants of the specific polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria, for example, as exemplified in the preceding paragraph. "Substantially the same function", as used herein, is intended to mean that the nucleic acid or polypeptide is capable of performing the function of the nucleic acid or polypeptide of which it is a variant. For example, a variant of an enzyme of the invention will be capable of catalyzing the same reaction as the enzyme. However, this should not be taken to mean that the variant has the same level of activity as the polypeptide or nucleic acid of which it is a variant.

One may assess whether a functionally equivalent variant has substantially the same function as the nucleic acid or polypeptide of which it is a variant using any number of known methods. However, by way of example, the methods described in Inui et al. (2008) may be used to assess enzymatic activity. "Overexpress", "overexpression" and similar terms and phrases, when used in connection with the invention, should be taken broadly to include any increase in the expression of one or more proteins when compared to the level of expression of the protein of a parent microorganism under the same conditions. It should not be taken to mean that the protein is expressed at any particular level.

A "parent microorganism" is a microorganism used to generate a recombinant microorganism of the present invention. The parent microorganism may be one that occurs in nature (i.e., a wild-type microorganism) or one that has been previously modified but which does not express or overexpresses one or more of the enzymes of the present invention. Accordingly, the recombinant microorganisms of the present invention have been modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parent microorganism.

The terms "nucleic acid constructs" or "vectors" and similar terms should be taken broadly to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle for transferring genetic material into a cell. The terms should be understood to include plasmids, viruses (including bacteriophages), cosmids, and artificial chromosomes. Constructs or vectors may include one or more regulatory elements, an origin of replication, a multiple cloning site, and/or a selectable marker, among other elements, sites, and markers. In a particular embodiment, the constructs or vectors are adapted to permit expression of one or more genes encoded by the construct or vector. Nucleic acid constructs or vectors include free nucleic acids as well as nucleic acids formulated with one or more agents to facilitate delivery to a cell (e.g., nucleic acid conjugated to a liposome, an organism in which the nucleic acid is contained).

It should be noted that the nucleic acids of the invention may be in any suitable form, including RNA, DNA or cDNA, including double-stranded and single-stranded nucleic acids.

In one aspect, the invention provides genetically modified microorganisms capable of using CO to produce 1-butanol and/or a precursor thereof as the main fermentation product. The microorganism is preferably an acetogenic recombinant microorganism that produces 1-butanol and/or a precursor thereof as the main fermentation product. In a particular embodiment, the acetogenic recombinant microorganism is capable of producing 1-butanol or a precursor thereof by fermentation of a substrate comprising CO at a concentration greater than approximately 1 mM or 0.075 g/L butanol per liter of fermentation broth.

In a particular embodiment, the microorganism comprises one or more exogenous nucleic acids adapted to express or overexpress one or more enzymes in the butanol biosynthesis pathway. In one embodiment, the microorganism is adapted to express one or more enzymes in the butanol biosynthesis pathway that are not naturally present in the parent microorganism from which it is derived or to overexpress one or more enzymes in the butanol biosynthesis pathway that are naturally present in the parent microorganism.

The microorganism may be adapted to express or overexpress one or more enzymes by any number of recombinant methods including, for example, increasing the expression of native genes within the microorganism (e.g., by introducing a strong or constitutive promoter to trigger expression of a gene), increasing the copy number of a gene encoding a particular enzyme by introducing exogenous nucleic acids encoding and adapted to express the enzyme, introducing an exogenous nucleic acid encoding and adapted not to express an enzyme naturally present in the parent microorganism.

In certain embodiments, the parent microorganism can be transformed to provide a combination of increased expression or overexpression of one or more genes native to the parent microorganism and introduction of one or more genes that are not native to the parent microorganism.

Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes selected from the group consisting of: thiolase; 3-hydroxybutyryl-CoA dehydrogenase; crotonase/crotonyl-CoA hydratase; butyryl-CoA dehydrogenase; Electron Transfer Flavoprotein A; and Electron Transfer Flavoprotein B. In one embodiment, the one or more nucleic acids encoding one or more enzymes are selected from nucleic acids of SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.

In one embodiment, the recombinant microorganism is adapted to express one or more of the genes which encode the enzymes thiolase (IUBMB enzyme nomenclature EC: 2.3.1.9) (thIA), 3-hydroxybutyryl-CoA dehydrogenase (EC: 1.1.1.157) (hbd), crotonase/crotonyl-CoA hydratase (EC: 1.1.1.157) (crt or cch) and/or butyryl-CoA dehydrogenase (EC4.2.1.55) (bed). In one embodiment, the microorganism is adapted to express all of these enzymes. In another embodiment, the genes which correspond to one or more of the nucleic acid sequences selected from SEQ ID NOs. 1 to 4 or functionally equivalent variants thereof. The recombinant microorganism of the invention may also contain two electron transfer proteins. In one embodiment, the electron transfer proteins are electron transfer flavoproteins (EC1.3.99.2) (etfAB) encoded by SEQ ID NOs. 5 and 6 or functionally equivalent variants thereof. The use of these electron transfer flavoproteins increases the efficiency of the microorganism in producing 1-butanol. The flavoproteins provide a stable complex that is necessary for Bed activity.

In a particular embodiment, the microorganism comprises one or more exogenous nucleic acids encoding each of thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, and Electron Transfer Flavoprotein B.

In one embodiment, the microorganism comprises a plasmid encoding one or more, or preferably each, of thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, and Electron Transfer Flavoprotein B.

In one embodiment or alternatively, the microorganism further comprises exogenous nucleic acids adapted to express one or more of the enzymes selected from the group consisting of: phosphotrans-butyrylase; butyrate kinase; ferric-redoxin-dependent aldehyde oxidoreductase (or in other words, aldehyde-reredoxin oxidoreductase); butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.

In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.

In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase. In particular embodiments, the microorganism comprises exogenous nucleic acids adapted to express each of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase.

In one embodiment, the microorganism comprises one or more nucleic acids suitable for expressing at least two of the enzymes in the 1-butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.

In one embodiment, the microorganism further comprises an acetate kinase/phosphotransacetylase promoter, although other promoters may be used. Preferably, the promoter corresponds to SEQ ID NO. 7 or a functionally equivalent variant thereof. Preferably, the promoter is contained in a construct encoding one or more of the enzymes mentioned hereinbefore.

Preferably, the parent microorganism is selected from the group of carboxytrophic acetogenic bacteria. In certain embodiments, the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium Ijungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

In a particular embodiment, the parent microorganism is selected from the group of ethanologenic, acetogenic Clostridia comprising the species C. autoethanogenum, C. Ijungdahlii, and C. ragsdalei and related isolates. These include, but are not limited to, strains of C. autoethanogenum JAI-1T (DSM10061) [Abrini J, Naveau H, Nyns E-J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351], C. autoethanogenum LBS1560 (DSM19630) [Simpson SD, Forster RL, Tran PT, Rowe MJ, Warner IL: Novel bacteria and methods thereof. International Patent 2009, WO/2009/064200], C. autoethanogenum LBS1561 (DSM23693), C. Ijungdahlii PETCT (DSM13528 = ATCC 55383) [Tanner RS, Miller LM, Yang D: Clostridium Ijungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232-236], C. Ijungdahlii ERI- 2 (ATCC 55380) [Gaddy JL: Clostridium stain which produces acetic acid from waste gases. US Patent 1997, 5,593,886], C. Ijungdahlii C-01 (ATCC 55988) [Gaddy JL, Clausen EC, Ko C-W: Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth. US Patent, 2002, 6,368,819], C. Ijungdahlii 0-52 (ATCC 55989) [Gaddy JL, Clausen EC, Ko C-W: Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth. US Patent, 2002, 6,368,819], C. ragsdalei P11T (ATCC BAA-622) [Huhnke RL, Lewis RS, Tanner RS: Isolation and Characterization of novel Clostridial Species. International Patent 2008, WO 2008/028055], related isolates such as "C. coskatii" [Zahn JA, Saxena J, Do Y, Patel M, Fishein S, Datta R, Tobey R: Clostridium coskatii, sp. nov., an Anaerobic Bacterium that Produces Ethanol from Synthesis Gas. Poster SIM Annual Meeting and Exhibition, San Francisco, 2010] or mutated strains such as C. Ijungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium Ijungdahlii. PhD thesis, North Carolina State University, 2010). These strains form a subgroup within the Clostridial rRNA cluster I and their 16s rRNA gene is more than 99% identical, with a similar low GC content of about 30%. However, DNA-DNA reassociation and DNA profiling experiments have shown that these strains belong to distinct species [Huhnke RL, Lewis RS, Tanner RS: Isolation and Characterization of novel Clostridial Species. International Patent 2008, WO 2008/028055],

All species in this group have similar morphology and size (logarithmically growing cells between 0.5-0.7 x 3-5 μm), are mesophilic (optimal growth temperature between 30-37 °C) and strictly anaerobic [Tanner RS, Miller LM, Yang D: Clostridium Ijung- dahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232-236; Abrini J, Naveau H, Nyns E-J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke RL, Lewis RS, Tanner RS: Isolation and Characterization of novel Clostridial Species. International Patent 2008, WO 2008/028055], Furthermore, they all share the same main phylogenetic traits, such as the same pH range (pH 4-7.5, with an initial pH optimum of 5.5-6), strong autotrophic growth on CO2-containing gases with similar growth rates and a similar metabolic profile, with ethanol and acetic acid as the main fermentation end products and small amounts of 2,3-butanediol and lactic acid formed under certain conditions [Tanner RS, Miller LM, Yang D: Clostridium Ijungdahlii sp. nov., an Acetogenic Speci- es in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232- 236; Abrini J, Naveau H, Nyns E-J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke RL, Lewis RS, Tanner RS: Isolation and Characterization of novel Clostridial Species. International Patent 2008, WO 2008/028055]. Indole production was observed with all three species. However, the species differ in substrate utilization of various sugars (e.g. rhamnose, arabinose), acids (e.g. gluconate, citrate), amino acids (e.g. arginine, histidine) or other substrates (e.g. betaine, butanol). Furthermore, some of the species were found to be auxotrophic for certain vitamins (e.g. thiamine, biotin), whereas others were not.

In one embodiment, the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, and butanol dehydrogenase, both before and after introduction of an exogenous nucleic acid.

In one embodiment, the microorganism produces butyraldehyde dehydrogenase and/or butanol dehydrogenase, both before and after introduction of an exogenous nucleic acid.

In a particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693.

In one embodiment, the recombinant microorganism of the invention has the characteristics that define the microorganism deposited in the DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) under accession number DSM24138.

The one or more exogenous nucleic acids may be delivered to a parent microorganism as free nucleic acids or may be formulated with one or more agents to facilitate the transformation process (e.g., nucleic acid conjugated to a liposome, an organism in which the nucleic acid is contained). The one or more nucleic acids may be DNA, RNA, or combinations thereof, as appropriate.

The microorganisms of the invention may be prepared from a parent microorganism and one or more exogenous nucleic acids using any number of methods known in the art for producing recombinant microorganisms. By way of example only, transformation (including transduction or transfection) may be achieved by electroporation, conjugation, or chemical and natural competence. Suitable transformation techniques are described, for example, in Sambrook et al., 1989.

In certain embodiments, because of the restriction systems which are active in the microorganism to be transformed, it is necessary to methylate the nucleic acid to be introduced into the microorganism. This can be done using a variety of techniques, including those described below and further exemplified in the Examples section hereinafter.

In another aspect, the invention provides a method of producing a recombinant microorganism comprising the following steps: a. introducing into a transport microorganism (i) an expression construct and (ii) a methylation construct comprising a methyltransferase gene; b. expressing the methyltransferase gene; c. isolating one or more constructs from the transport microorganism; and d. introducing one or more constructs into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target organism.

In one embodiment, the step b methyltransferase gene is constitutively expressed. In another embodiment, expression of the step b methyltransferase gene is induced.

The microorganism is a transport microorganism, preferably a restriction-negative microorganism that facilitates methylation of the nucleic acid sequences that comprise the expression construct. In a particular embodiment, the transport microorganism is a restriction-negative E. coli, Bacillus subtillis or Lactococcus lactis.

Once the expression construct and the methylation construct are introduced into the transport microorganism, the methyltransferase gene present in the methylation construct is expressed. In one embodiment, where expression is to be induced, induction may be through any suitable promoter system although, in a particular embodiment of the invention, the methylation construct comprises an inducible lac promoter (preferably encoded by SEQ ID NO. 28) and is induced upon the addition of lactose or an analogue thereof, most preferably isopropyl-β-thio-D-galactoside (IPTG). Other suitable promoters include ara, teta or the T7 system. In an alternative embodiment of the invention, the promoter of the methylation construct is a constitutive promoter.

In one embodiment, the promoter of the expression construct is a constitutive promoter that is preferably highly active under suitable fermentation conditions. However, an inducible promoter may be used. In preferred embodiments, the promoter of the expression construct is selected from the group comprising the promoter of the phosphate butyryl/acetyl transferase operon, pyruvate:ferredoxin oxidoreductase (SEQ ID NO. 48), the Wood-Ljungdahl gene cluster (SEQ ID NO. 47), the Rnf operon (SEQ ID NO. 49) or the ATP synthase operon (SEQ ID NO. 50). Preferably, the promoter of the phosphotransacetylase/acetate kinase operon corresponds to SEQ ID NO. NO. 7 or a functionally equivalent variant thereof. Figure 8 shows that expression of genes operatively linked to these promoters have a high level of expression in Clostridium autoethanogenum under standard conditions.

In a particular embodiment, the methylation construct has an origin of replication specific to the identity of the transport microorganism, such that any genes present in the methylation construct are expressed in the transport microorganism. Preferably, the expression construct has an origin of replication specific to the identity of the target microorganism, such that any genes present in the expression construct are expressed in the target microorganism.

Expression of the methyltransferase enzyme results in methylation of the genes present in the expression construct. The expression construct can then be isolated from the transport microorganism according to any of a number of known methods. By way of example only, the methodology described in the Examples section below can be used to isolate the expression construct.

In a particular embodiment, both constructs are simultaneously isolated. The expression construct may be introduced into the target microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section below may be used. Once the expression construct is methylated, the nucleic acid sequences present in the expression construct may be incorporated into the target microorganism and successfully expressed.

In another embodiment, the invention provides a method of producing a recombinant microorganism comprising: a. methylating an expression construct in vitro by a methyltransferase, preferably according to SEQ ID NO. 28 or a functionally equivalent variant thereof; and b. introducing an expression construct, preferably according to the fifth aspect, into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

It is contemplated that a methyltransferase gene of the invention, preferably according to SEQ ID NO. 27 or a functionally equivalent variant thereof, can be introduced into a carrier microorganism and overexpressed. The resulting methyltransferase enzyme can be harvested using known methods and used in vitro to methylate an expression construct, preferably the expression construct is as defined in the fifth aspect. The expression construct can then be introduced into the target microorganism for expression. Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the major fermentation product.

In another embodiment, the invention provides a method of producing a recombinant microorganism comprising: a. introducing into the genome of a transport microorganism a methyltransferase gene, preferably according to SEQ ID NO. 27 or a functionally equivalent variant thereof; b. introducing an expression construct into the transport microorganism; c. isolating one or more constructs from the transport microorganism; and d. introducing at least one expression construct into a target microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the target microorganism.

Standard methods are used for introducing a methyltransferase gene, preferably according to SEQ ID NO. 27, into the genome of the transport microorganism. The methyltransferase may be constitutively expressed by the microorganism and result in the production of a methyltransferase enzyme, preferably according to SEQ ID NO. 28 or a functionally equivalent variant thereof. An expression construct is methylated, isolated and introduced into the target microorganism which preferably produces 1-butanol and/or a precursor thereof as the major fermentation product.

The invention also includes microorganisms comprising a recombinant methyltransferase gene or methylation construct as described herein.

The present invention also provides a hybrid methyltransferase gene (SEQ ID NO. 28) developed after analysis of nucleic acid sequences of methyltransferase and restriction barrier systems of C. autoethanogenum, C. Ijungdahlii and C. ragsdalei.

The methyltransferase gene is expressed in a transport microorganism, which results in the production of a methyltransferase enzyme that methylates the sequence of the expression construct. The methyltransferase gene may be present in a construct or integrated into the genome of the transport microorganism. The hybrid methyltransferase gene is codon optimized for E. coli and may be incorporated into a methylation construct (Figure 5). The methyltransferase gene may be codon optimized for use in another species of microorganism if appropriate, e.g., Bacillus subtilis. Methods for codon optimization are standard and will be known to those of skill in the art (Carbone et al., 2003). Also incorporated within the scope of the invention are methyltransferase genes that have at least 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or more nucleic acid sequence identity to SEQ ID NO. 28 and express a polypeptide that is capable of methylating DNA.

It will be appreciated by those skilled in the art that the method of methylating the methyltransferase gene will have utility in a variety of microorganisms. In one embodiment, the target microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium Ijungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi. In a particular embodiment, the target microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium Ijungdahlii, and Clostridium ragsdalei. In a particular embodiment, the target microorganism is Clostridium autoethanogenum DSM23693.

The invention also provides various nucleic acids or nucleic acid constructs as described in aspects 4, 5, 14, 15, 16, 18, 19 and 21 of the invention described hereinbefore.

In another embodiment of the invention, there is an expression construct comprising one or more nucleic acids encoding one or more enzymes chosen from thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, butyryl-CoA dehydrogenase, and an electron transfer protein or a functionally equivalent variant thereof. Preferably, the electron transfer protein is Electron Transfer Flavoprotein A or Electron Transfer Flavoprotein B. In a particular embodiment, both Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B are included in the expression construct.

Exemplary sequence information for each gene and equivalent enzyme is provided in GenBank, as detailed in Table 1 hereinafter. Those skilled in the art will readily appreciate alternative genes and enzymes which may be used. In one embodiment, the enzymes are encoded by nucleic acid SEQ ID NO. 1 to 6, which may be present in any order in the construct or in the order shown in Figure 2. SEQ ID NO. 8 to 13 and SEQ ID NO. 16 to 23 are novel sequences used to clone and sequence the genes mentioned in the immediately preceding paragraph.

In order to obtain 1-butanol from a precursor, activity of one or more of butyraldehyde dehydrogenase (EC 1.2.1.10), alcohol dehydrogenase (EC 1.1.1.1), phosphotransbutyrylase (EC 2.3.1.19), butyrate kinase (EC 2.7.2.7), aldehyde-reductoxin oxidoreductase (EC1.2.7.5) and alcohol dehydrogenase (EC 1.1.1.1):2,7 may be required. The alcohol dehydrogenase of the invention is a butanol dehydrogenase. In certain embodiments, butyraldehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) or phosphotransbutyrylase (EC 2.3.1.19), butyrate kinase (EC 2.7.2.7), aldehyde ferredoxin oxidoreductase (EC 1.2.7.5) and alcohol dehydrogenase (EC 1.1.1.1) or a combination of both sets of enzymes is required. In one embodiment, the butyraldehyde dehydrogenase and butanol dehydrogenase activity is conferred by a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. These various enzymes are shown in the butanol biosynthesis pathway illustrated in Figure 1. In some microorganisms, butyraldehyde dehydrogenase, butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase, and/or aldehyde:ferredoxin oxidoreductase are naturally expressed by the microorganism and therefore catalyze the conversion of butyryl-CoA to 1-butanol.

Accordingly, in one embodiment, the expression construct comprises nucleic acids encoding one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase, in addition to or alternatively to one or more of thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, and an electron transfer protein.

Examples of suitable enzymes and amino acids and nucleic acid sequence information include, but are not limited to: butyraldehyde dehydrogenase, such as Aid from C. beijerinckii (ABR35947, Gl: 149905114), C. saccharobutylicum (CAQ57983, Gl: 189310620) or Clostridium saccharoperbutylacetoniucm (AAP42563, Gl: 31075383); butanol dehydrogenase, such as BdhB from C. acetobutylicum (NP_349891, Gl: 15896542); a butyraldehyde/butanol dehydrogenase enzyme, such as AdhE1 from C. acetobutylicum (NP_149325, Gl: 15004865) or AdhE2 from C. acetobutylicum (NP_149199, Gl: 15004739), C. beijerinckii (YP_001307449, Gl: 150015195); a phosphotransbutyrylase, such as Ptb from C. acetobutylicum (NP_348368); a butyrate kinase, such as Buk from C. acetobutylicum (AAK81015.1); aldehyde:ferredoxin oxidoreductase AOR from C. acetobutylicum (NP_348637). Those skilled in the art to which the invention relates can readily appreciate alternative examples of enzymes suitable for use in the invention. The inventors have also identified a number of novel enzymes and genes that can be used in the invention, details of which are provided hereinafter in the Examples section (in particular, see Tables 7 and 10). The invention also encompasses functionally equivalent variants of these enzymes and genes and their use in methods of the invention.

Inclusion of one or more of these genes may help to avoid butyrate co-production altogether, increasing the efficiency of 1-butanol production. The invention also provides recombinant microorganisms comprising one or more nucleic acids adapted to express or enhance the expression of one or more of these enzymes.

In one embodiment, the nucleic acid(s) encode(s) an enzyme chosen from the group of enzymes listed in Tables 7 through 10 hereinafter and functional equivalents of any one or more thereof. In a particular embodiment, the nucleic acids are chosen from the group of nucleic acids in Tables 7 through 10 hereinafter and functional equivalents of any one or more thereof.

In one embodiment, the expression construct encodes at least two enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.

Preferably, the expression construct further comprises a suitable promoter as described hereinbefore. In one embodiment, the promoter is a butyryl/acetyl phosphate transferase promoter. Preferably, the promoter corresponds to SEQ ID NO. 7 or a functionally equivalent variant thereof.

In a preferred embodiment, the expression construct comprises a nucleic acid encoding all of said enzymes. It will be appreciated by those skilled in the art that the expression construct may comprise nucleic acids encoding alternative electron transfer proteins.

The genes to be expressed in the recombinant microorganism may be assembled into the expression construct under the control of any suitable promoter. In a particular embodiment, the promoter allows substantially constitutive expression of the genes under its control. In a particular embodiment, the promoter is a butyryl/acetyl phosphate transferase promoter (SEQ ID NO. 7). Other promoters that may find use in the present invention include those from C. autoethanogenum (or C. Ijungdahlii). The inventors have also identified a number of other promoters that are operably linked to genes that were highly expressed under typical fermentation conditions in Clostridium autoethanogenum (Figure 8). Analysis of gene expression under over 200 typical fermentation conditions using real-time PCR identified a number of suitable promoters. These include pyruvate oxidoreductase (SEQ ID NO. 48), the Wood-Ljungdahl gene cluster (SEQ ID NO. 47), Rnf operon (SEQ ID NO. 49) and ATP synthase operon (SEQ ID NO. 50). It will be appreciated by those skilled in the art that other promoters which can direct expression, preferably a high level of expression under appropriate fermentation conditions, will be effective as alternatives to the presently preferred embodiments.

In one embodiment, the invention comprises a construct, recombinant microorganism, or nucleic acid sequence comprising nucleic acid SEQ ID NOs. 1 to 6 in the order shown in Figure 2. However, it will be appreciated by those skilled in the art that the invention may still have the desired utility when the nucleic acid sequences are presented in any order and with one or more of the sequences missing.

In another embodiment, the invention comprises a nucleic acid comprising the promoter sequence represented by SEQ ID NO. 7 or a functionally equivalent variant thereof, the construct comprising said promoter and recombinant microorganisms comprising the same.

It will be appreciated that an expression construct of the present invention may contain any number of regulatory elements in addition to the promoter, as well as additional genes suitable for expression of other proteins, if desired. In one embodiment, the construct comprises one promoter. In another embodiment, the construct comprises two or more promoters. In a particular embodiment, the construct includes one promoter for each gene to be expressed. In one embodiment, the construct comprises one or more ribosomal binding sites, preferably one ribosomal binding site for each gene to be expressed.

It will be appreciated by those skilled in the art that the nucleic acid sequences and construct sequences defined herein may contain conventional linker nucleotides, such as those required for ribosomal binding sites and/or restriction sites. Such linker sequences are not to be construed as being required and do not constitute a limitation on the defined sequences.

When the expression construct of the present invention is expressed in an acetogenic microorganism, the microorganism produces 1-butanol or a precursor thereof as the major fermentation product. It is contemplated that other genes encoding enzymes that catalyze various steps of the butanol or Wood-Ljungdahl biosynthesis pathways may also be incorporated into the expression construct so as to produce 1-butanol as the major fermentation product.

It is contemplated that the expression construct and the methylation construct, as defined above, may be combined to provide a composition of matter. Such a composition has particular utility in circumventing restriction barrier mechanisms in a wide variety of microorganisms, but in a preferred embodiment, the recombinant microorganism produced using the composition produces 1-butanol or a precursor thereof as the major fermentation product.

Nucleic acids and nucleic acid constructs, including expression constructs, of the present invention may be constructed using any number of standard methods in the art. For example, chemical synthesis or recombinant techniques may be used. Such techniques are described, for example, in Sambrook et al. (1989). Other exemplary techniques are described in the Examples section hereinafter. Essentially, the individual genes and regulatory elements will be operably linked to each other so that the genes can be expressed to form the desired proteins. Vectors suitable for use in the present invention will be appreciated by those skilled in the art. However, by way of example, the following vectors may be suitable: shuttle vectors pMTL80000, plMP1, and pJIR750, and the plasmids exemplified in the Examples section hereinafter.

To the extent that the invention provides novel nucleic acids and nucleic acid vectors, it also provides nucleic acids which are capable of hybridizing to at least a portion of a nucleic acid described herein, a nucleic acid complementary to any of them, or a functionally equivalent variant of any of them. Such nucleic acids will preferably hybridize to such nucleic acids, a nucleic acid complementary to any of them, or a functionally equivalent variant of any of them under stringent hybridization conditions. "Stringent hybridization conditions" means that the nucleic acid is capable of hybridizing to a target template under conventional hybridization conditions, such as those described in Sambrook et al. (1989). It will be appreciated that the minimum size of such nucleic acids is a size which is capable of forming a stable hybrid between a given nucleic acid and the complementary sequence to which it is desired to hybridize. Accordingly, the size is dependent on the nucleic acid composition and percent homology between the nucleic acid and its complementary sequence, as well as the hybridization conditions that are used (e.g., temperature and salt concentration). In one embodiment, the nucleic acid is at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, or at least 30 nucleotides in length.

It should be noted that the nucleic acids of the invention may be in any suitable form, including RNA, DNA or cDNA, including double-stranded and single-stranded nucleic acids.

The invention also provides host organisms, particularly microorganisms, including viruses and bacteria and yeasts, comprising any one or more of the nucleic acids described herein.

The invention provides a method of producing 1-butanol and/or a precursor thereof via microbial fermentation comprising fermentation of a gaseous substrate comprising CO using a recombinant microorganism. In certain embodiments, 1-butanol or a precursor thereof is co-produced with another fermentation product (e.g., ethanol). In one embodiment, 1-butanol or a precursor thereof is the primary fermentation product. In one embodiment, the recombinant microorganism is as previously described herein.

In one embodiment, 1-butanol and/or a precursor thereof is produced in a yield of approximately 0.075 g per liter of fermentation broth (g/L) to approximately 20 g/L. In one embodiment, the yield is approximately 0.15 g/L to approximately 1.54 g/L. In other embodiments, the yield is approximately 10 g/L, approximately 5 g/L, or approximately 2 g/L. Preferably, the yield of 1-butanol is up to the limit at which butanol becomes toxic to bacteria.

Preferably, the fermentation comprises the steps of anaerobically fermenting a substrate in a bioreactor for the production of 1-butanol and/or a precursor thereof using recombinant microorganisms as described herein.

Where the 1-butanol precursor is referred to herein, it is contemplated that it may be optionally converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase, a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase and/or ferredoxin-dependent aldehyde oxidoreductase. Preferably, the microorganism produces one or more of these enzymes, either before or after introduction of a recombinant nucleic acid.

In one embodiment of the invention, the gaseous substrate fermented by the microorganism is a gaseous substrate containing CO. The gaseous substrate may be a CO-containing waste gas obtained as a byproduct of an industrial process or from another source, such as from automobile exhaust. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal product manufacturing, such as steel milling, nonferrous product manufacturing, petroleum refining processes, coal gasification, electrical power production, carbon black production, ammonia production, methanol production, and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial process prior to emission to the atmosphere using any convenient method. The CO may be a component of syngas (gas comprising carbon monoxide and hydrogen). CO produced from industrial processes is normally burned to produce CO2 and therefore the invention has particular utility in reducing CO2 greenhouse gas emissions and producing butanol for use as a biofuel. Depending on the composition of the CO-containing gaseous substrate, it may also be desirable to treat it to remove any undesirable impurities, such as dust particles, prior to introducing it into the fermentation process. For example, the gaseous substrate may be filtered or purified using known methods.

It will be appreciated that in order for growth of the bacteria and fermentation of CO to 1-butanol to occur, in addition to the gaseous substrate containing CO, a suitable liquid nutrient medium must be fed to the bioreactor. A nutrient medium will contain sufficient vitamins and minerals to allow growth of the microorganism used. Suitable media for anaerobic fermentation for butanol production using CO are known in the art. For example, suitable media are described in Biebel (2001). In one embodiment of the invention, the medium is as described in the Examples section hereinafter.

Fermentation should desirably be carried out under conditions suitable for fermentation of CO to butanol to occur. Reaction conditions that must be considered include pressure, temperature, gas flow rate, liquid flow rate, pH of the medium, redox potential of the medium, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gaseous substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.

Furthermore, it is often desirable to increase the CO concentration of a substrate stream (or partial pressure of CO in a gaseous substrate) and thus increase the efficiency of fermentation reactions where CO is a substrate. Operation at higher pressures allows a significant increase in the rate of transfer of CO from the gas phase to the liquid phase, where it can be taken up by the microorganism as a carbon source for butanol production. This, in turn, means that the retention time (defined as the volume of liquid in the bioreactor divided by the inlet gas flow rate) can be reduced when bioreactors are maintained at elevated pressure, rather than at atmospheric pressure. The optimum reaction conditions will depend, in part, on the particular microorganism of the invention used. However, in general, it is preferred that fermentation be carried out at a pressure greater than ambient pressure. Furthermore, since a given rate of CO to butanol conversion is in part a function of substrate retention time, and achieving a desired retention time in turn determines the required bioreactor volume, the use of pressurized systems can significantly reduce the required bioreactor volume and, consequently, the capital cost of fermentation equipment. According to the examples provided in U.S. Patent No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e., bioreactors operated at 10 atmospheres of pressure require only one-tenth the volume of those operated at 1 atmosphere of pressure.

The benefits of conducting gas-to-ethanol fermentation at elevated pressures have been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations conducted at pressures of 30 psi and 75 psi, yielding ethanol productivities of 150 g/L/day and 369 g/L/day, respectively. However, exemplary fermentations conducted using similar media and input gas compositions at atmospheric pressure have been found to produce between 10 and 20 times less ethanol per liter per day.

The composition of the gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, O2 can decrease the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases at stages of a fermentation process before or after fermentation can increase the load on those stages (e.g., where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in fermentation). Consequently, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove undesirable components and increase the concentration of desirable components.

In certain embodiments, a culture of a bacterium of the invention is maintained in an aqueous culture medium. Preferably, the aqueous culture medium is a minimal anaerobic microbial growth medium.

Suitable means are known in the art and described, for example, in US Patent Nos. 5,173,429 and 5,593,886 and WO 02/08438 and as described in the Examples section hereinafter.

Butanol or a mixed alcohol stream containing butanol and one or more other alcohols can be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, and extractive fermentation including, for example, liquid-liquid extraction. By-products, such as acids, including butyrate, can also be recovered from the fermentation broth using methods known in the art. For example, an adsorption system involving an activated carbon filter or electrodialysis can be used. Alternatively, continuous gas stripping can also be used.

In certain preferred embodiments of the invention, butanol and by-products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering butanol and, optionally, acid from the broth. Alcohols may conveniently be recovered, for example, by distillation, and acids may be recovered, for example, by adsorption onto activated carbon. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell-free permeate remaining after the alcohol(s) and acid(s) are removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor.

Also, if the pH of the broth was adjusted as described above to increase acetic acid adsorption to activated carbon, the pH should be adjusted again to a pH similar to that of the broth in the fermentation bioreactor before it is returned to the bioreactor.

In one embodiment of the invention, butanol is recovered from the fermentation reaction using extractive fermentation procedures in which butanol is recovered in an oil phase in the reactor. Those skilled in the art will readily appreciate techniques for achieving this.

EXAMPLES

The invention will now be described in greater detail with reference to the following non-limiting examples.

Genetic modifications were performed using a plasmid containing a synthetic operon consisting of a strong native promoter from C. autoethanogenum that controls genes for thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, and two electron transfer flavoproteins from C. acetobutylicum (Fig. 1-2). This plasmid was methylated in vivo using a novel methyltransferase and then transformed into C. autoethanogenum DSM23693. Production of 1-butanol as the major fermentation product was shown in different industrial gas streams (steel mill off-gases, syngas).

Expression Plasmid Construct:

Standard molecular cloning and recombinant DNA techniques were used in the present invention and are described by Sambrook et al., 1989 and Ausubel et al., 1987. DNA sequences of butanol biosynthesis genes from Clostridium acetobutylicum ATCC824 used were obtained from NCBI (Table 1). The promoter of the acetate kinase/phosphotransacetylase operon from C. autoethanogenum DSM10061 was sequenced and used for expression of target genes (Table 1). RT-PCR experiments showed that this promoter is constitutively expressed at a high level (Figure 8). Table 1: Sources of 1-butanol Pathway Genes Genomic DNA from Clostridium acetobutylicum ATCC824 and Clostridum autoethanogenum DSM10061 was isolated using a method modified by Bertram and Dürre (1989). A 100-ml overnight culture was harvested (6,000 × g, 15 min, 4 °C), washed with potassium phosphate buffer (10 mM, pH 7.5), and suspended in 1.9 ml of STE buffer (1 mM EDTA, 50 mM Tris-HCl, 1 mM, 200 mM sucrose, pH 8.0). 300 μl of lysozyme (~100,000 U) was added and the mixture was incubated at 37 °C for 30 min, followed by the addition of 280 μl of a 10% (w/v) SDS solution and another incubation for 10 min. RNA was digested at room temperature by the addition of 240 μl of an EDTA solution (0.5 M, pH 8), 20 μl of Tris-HCl (1 M, pH 7.5) and 10 μl of RNase A (Fermentas). Then, 100 μl of proteinase K (0.5 L) was added and proteolysis was carried out for 1–3 h at 37 °C. Finally, 600 μl of sodium perchlorate (5 M) were added, followed by a phenol-chloroform extraction and isopropanol precipitation. The quantity and quality of DNA were verified by spectrophotometry.

Genes for butanol biosynthesis and the acetate kinase/phosphotransacetylase promoter were amplified by PCR with the oligonucleotides in Table 2 using iProof High Fidelity DNA polymerase (Bio-Rad Laboratories) and the following program: initial denaturation at 98°C for 30 seconds, followed by 32 cycles of denaturation (98°C for 10 seconds), annealing (50-62°C for 30-120 seconds), and elongation (72°C for 45 seconds) before a final extension step (72°C for 10 minutes). Table 2: Oligonucleotides for Cloning

The amplified 498 bp promoter region of the acetate kinase/phosphotransacetylase (Ppta-ack) operon was cloned into the E. coli-Clostridium shuttle vector pMTL 85141 (SEQ ID 14; FJ797651.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using Not\ and Nde\ restriction sites and the DH5α-T1R strain (Invitrogen). The plasmid pMTL85145 and the 1,194 bp PCR product of the thiolase gene were both cut with Nde\ and EcoRI. The ligation was transformed into E. coli XL1-Blue MRF' Kan (S-tratagene), resulting in plasmid pMTL85145-thlA. Subsequently, the PCR-amplified 4,764-bp fragment of the crt-bcd-etfB-etfA-hbd operon from C. acetobutylicum ATCC 824 was cloned into this vector using KpnI and Ba- mHI and E. coli ABLE K (Stratagene), creating plasmid pMTL85145-thlA-crt-hbd. Finally, the antibiotic resistance cassette was changed from chloramphenicol to clarithromycin. Therefore, an ermB cassette was released from vector pMTL82254 (SEQ ID 15; FJ797646.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using restriction enzymes PmeI and FseI and exchanged for a catP cassette from plasmid pMTL85145-thlA-crt-hbd. The resulting expression plasmid insert, pMTL85245-thlA-crt-hbd (SEQ ID NO. 31), was completely sequenced using the oligonucleotides provided in Table 3 and the results confirmed that the butanol biosynthesis genes were free of mutations (Figure 3). Table 3: Oligonucleotides for Sequencing

DNA methylation:

A hybrid methyltransferase gene fused to an inducible lac promoter was designed (SEQ ID NO. 28) by aligning methyltransferase genes from C. autoethanogenum (SEQ ID NO. 24), C. Ijungdahlii (SEQ ID NO. 25), and C. ragsdalei (SEQ ID NO. 26) (Figures 4a, 4b, and 4c). Expression of the methyltransferase gene resulted in the production of a methyltransferase enzyme according to SEQ ID NO. 28. Amino acid sequence alignment data of methyltransferase are shown in Figure 4d. The hybrid methyltransferase gene (SEQ ID NO. 27) was chemically synthesized and cloned into the pGS20 vector (SEQ ID 29; ATG:biosynthetics GmbH, Merzhausen, Germany) using EcoRI (Fig. 5). The resulting methylation plasmid pGS20-methyltransferase was double transformed with the expression plasmid pMTL85245-thlA-crt-hbd into restriction-negative E. co//XL1-Blue MRF' Kan (Stratagene). In vivo methylation was induced by the addition of 1 mM IPTG, and methylated plasmids were isolated using the PureLink™ HiPure Plasmid Maxiprep kit (Invitrogen). The resulting methylated plasmid composition was used for transformation of C. autoethanogenum DSM23693.

Transformation:

During the complete transformation experiment, C. autoethanogenum DSM23693 and C. Ijundahlii (DSM 13528) were grown in PETC medium (Tab. 4) with 10 g/L fructose and 30 °C steel mill waste gas (collected from the New Zealand Steel Site in Glenbrook, NZ; composition: 44% CO, 32% N2, 22% CO2, and 2% H2) as carbon source at 37 °C using standard anaerobic techniques described by Hungate (1969) and Wolfe (1971). Table 4: PETC medium (ATCC 1754 medium; http://www.atcc.org/Attachments/2940.pdf)

To make competent cells, a 50 ml culture of C. autoethanogenum DSM23693 and a 50 ml culture of C. Ijundahlii 5 DSM13528 were subcultured in fresh medium for 3 consecutive days.

These cells were used to inoculate 50 ml of PETC medium containing 40 mM DL-threonine at an OD600 nm of 0.05. When the culture reached an OD600 nm of 0.4, the cells were transferred to an anaerobic chamber and harvested at 4,700 × g and 4 °C. The culture was washed twice with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl, 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 μl of fresh electroporation buffer. This mixture was transferred to a precooled electroporation cuvette with a 0.4 cm electrode gap containing 1 μg of the methylated plasmid mixture (and, in the case of C. ijundahlii, 1 μl of Type 1 Restriction Inhibitor (Epicentre Biotechnologies)) and immediately conditioned using a Gene Pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 Q, and 25 μF. Time constants of 3.7–4.0 ms were obtained. The culture was transferred to 5 ml of fresh medium. Cell regeneration was monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, cells began to grow again. Once the biomass had doubled from this point, cells were harvested, suspended in 200 μl of fresh medium, and cultured on selective PETC plates (containing 1.2% Bacto™ agar (BD)) with 4 μg/μl Clarithromycin. After 4–5 days of inoculation with steel mill gas at 30 psi at 37 °C, 15–80 colonies per plate were clearly visible.

The colonies were used to inoculate 2 ml of PETC medium containing 4 μg/mL Clarithromycin. When growth occurred, the culture was scaled up to 5 ml and then 50 ml of PETC medium containing 4 μg/mL Clarithromycin and 30 psi steel mill gas as the sole carbon source.

Confirmation of Successful Transformation:

C. autoethanogenum: To check DNA transfer, a plasmid miniprep was performed from a 10 ml culture volume using the QIAprep Spin Miniprep kit (Qiagen). Due to the Clostridial exonuclease activity (Burchhardt and Dürre, 1990), the plasmid DNA isolated from 4 clones analyzed was partially degraded and resulted in only a spot on an agarose gel, whereas the isolation of plasmids from the original C. autoethanogenum DSM23693 strain did not result in an overall signal (Fig. 6). However, the quality of the isolated plasmid DNA was sufficient to perform a control PCR using four primer sets covering all the different relevant regions of the plasmid (Table 5). PCR was performed with Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) using standard conditions (95 °C for 5 min; 32 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; 72 °C for 10 min). PCR of all four transformants analyzed resulted in the same signals as the original methylated plasmid mixture as template (Fig. 6). As an additional control, 1 μl of each of the partially degraded isolated plasmids was re-transformed into E. coli//XL1-Blue MRF' Kan (Stratagene), from which the plasmids could be cleanly isolated and verified by restriction digests.

To confirm the identity of the four clones, genomic DNA was isolated (see above) from 40 ml of each culture and a PCR was performed against the 16S rRNA gene (Table 5; Weisberg et al., 1991) using Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) and standard conditions (95 °C for 5 min; 32 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; 72 °C for 10 min). The respective PCR products were purified and sequenced. The sequences of all clones showed at least 99.9% identity to the C. autoethanogenum 16S rRNA gene (SEQ ID 30; Y18178, Gl: 7271109).

The respective strain was deposited at the DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) under accession number DSM24138 on October 26, 2010. C. Ijungdahlii-. Clostridium Ijungdahlii transformants were confirmed using the same method and primer sets. Sequencing of the 16S rRNA gene resulted in a 100% match to the 16S gene of Clostridium Ijungdahlii (SEQ ID 119; CP001666, Gl: 300433347). Table 5: Oligonucleotides for PCR Confirmation of Plasmid and Species

Production of 1-butanol:

To demonstrate the production of 1-butanol from CO as the sole carbon and energy source, PETC medium lacking yeast extract and fructose was prepared and inoculated with the novel strains of C. autoethanogenum and C. Ijungdahlii harboring the butanol plasmid pMTL85245-thlA-crt-hbd. Bottles were pressurized with 30 psi of a gas stream containing CO from two industrial sources, steel mill off-gas (collected from the New Zealand Steel Site in Glenbrook, NZ; composition: 44% CO, 32% N2, 22% CO2, 2% H2) and syngas (Range Fuels Inc., Broomfield, CO; composition: 29% CO, 45% H2, 13% CH4, 12% CO2, 1% N2). Production of 1-butanol could be demonstrated with both strains and both gas mixtures over several subculture periods. Co-production of butyrate was also observed. Neither 1-butanol nor butyrate was detected in samples of unmodified strains of C. autoethanogenum DSM23693 and C. Ijungdahlii DSM13528 under the same conditions.

Metabolite analysis was performed by HPLC using an Agilent 1100 Series HPLC system equipped with a Refractive Index Detector (RID) operated at 35 °C and an Alltech IOA-2000 organic acid column (150 × 6.5 mm, 5 μm particle size) maintained at 60 °C. Slightly acidified water (0.005 M H2SO4) was used as the mobile phase with a flow rate of 0.7 ml/min. To eliminate proteins and other cellular debris, 400 μl of samples were mixed with 100 μl of a 2% (w/v) 5-sulfosalicylic acid solution and centrifuged at 14,000 × g for 3 min to separate precipitated debris. 10 μl of the supernatant was then injected into the HPLC for analysis.

In serum bottle experiments, the highest 1-butanol production was observed in two static cultures of C. autoethanogenum harboring the butanol plasmid pMTL85245-thlA-crt-hbd. In these cultures, 1-butanol was the major fermentation end product observed, at 1.54 g/L (25.66 mM) (Table 6, Fig. 7). The production of other metabolites was reduced compared with the parent C. autoethanogenum strain DSM23693, which produced only ethanol, acetate, and 2,3-butanediol. Although carbon flux was diverted to 1-butanol production, the total amount of carbon incorporated into the metabolic end products remained virtually the same (Table 6). The slight increase of 20% is likely the result of extra discharge of reduction equivalents for the production of 1-butanol and butyrate compared to ethanol and acetate, respectively. The production of 2,3-butanediol, which usually acts as an electron sink, was completely reduced. Table 6: Metabolite Production and Carbon Balance of C. autoethanogenum Harboring the Butanol Plasmid pM-TL85245-thlA-crt-hbd Compared with C. autoethanogenum DSM23693 Original

Production of 1-butanol was also observed in cultures of C. Ijungdahlii DSM13528 harboring the butanol plasmid pMTL85245-thlA-crt-hbd in significant amounts up to 0.36 g/L (6 mM), although lower than that of C. autoethanogenum DSM23693 harboring the same plasmid. This could be explained because C. autoethanogenum DSM23693 is a strain with enhanced alcohol production and, correspondingly, the unmodified strain of C. autoethanogenum DSM23693 produces more ethanol and less acetate than the unmodified strain of C. Ijungdahlii DSM13528 (both strains produce neither butanol nor butyrate). C. Ijungdahlii harboring the butanol plasmid pMTL85245-thlA-CRT-hbd had a higher 1-butanol:butyrate ratio than C. autoethanogenum. The ratio of 1-butanol to butyrate, however, can be altered by process conditions. This allows the production of 1-butanol as the major fermentation product but also the production of butyrate as the major fermentation product in both C. autoethanogenum and C. Ijungdahlii strains. In serum vial experiments, 1-butanol:butyrate molar ratios ranging from 50:1 to 1:30 were observed with C. autoethanogenum and 20:1–1:30 with C. Ijungdahlii. Cultures which were incubated under agitation generally produced higher levels of butyrate and lower levels of 1-butanol compared with static cultures. The concentration of CO (and H2) in the headspace was found to have an effect on the 1-butanol:butyrate ratio as well. In cultures with less CO in the headspace, butyrate production was more favored and it could be produced as the major fermentation product. Correspondingly, higher titers of 1-butanol were observed with the CO-rich steelmaking gas (44% CO) than with the CO-poor syngas (29% CO) in serum flask experiments. A maximum of 1.08 g/L (12.8 mM) butyrate was observed with Clostridium autoethanogenum harboring a pMTL85245-thlA-crt-hbd plasmid and a level of 1.03 g/L (12.5 mM) with C. Ijungdahlii harboring the same plasmid. This effect can be explained by the extra carbon in the system and also the additional reducing power generated from the oxidation of CO by carbon monoxide dehydrogenase (CODH).

Conversion of Butyryl-CoA to Butyrate and Butanol:

The expression plasmid contains only the genes required for the production of butyryl-CoA from acetyl-CoA. Butyryl-CoA can then be converted directly to butanol by the action of a butyraldehyde dehydrogenase and butanol dehydrogenase (Fig. 1). A second possibility is that butyryl-CoA is converted to butyrate via a phosphotransbutyrylase and butyrate kinase (Fig. 1), in which case ATP is obtained via substrate level phosphorylation (SLP). Since the operation of the Wood-Ljungdahl pathway requires ATP, acetogenic cells depend on ATP from SLP, which is also reflected in the fact that all known acetogenic bacteria produce acetate (Drake et al., 2006). However, the recombinant cell can now also generate ATP via SLP also by producing butyrate. Butyrate can then be further reduced to butyraldehyde via an aldehyde:ferredoxin oxidoreductase (RPA) (Fig. 1). This reaction can be triggered by reduced ferredoxin provided by the oxidation of CO via carbon monoxide dehydrogenase (CO + Fdred -> CO2 + Fdox), the initial step in the Wood-Ljungdahl pathway. Butyraldehyde can then be converted to butanol via a butanol dehydrogenase (Fig. 1). Conversion of externally added butyrate to butanol by a culture of C. autoethanogenum has been demonstrated (W02009/113878).

Respective genes/enzymes with butyraldehyde dehydrogenase, butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase and aldehyde-redoxin oxidoreductase activities were identified by the inventors in C. autoethanogenum, C. Ijungdahlii and C. ragsdalei (Tab. 7–10). Potential genes and enzymes were predicted by comparison with characterized genes and enzymes using BLAST (Altschul et al., 1990), COG (Tatusov et al., 2003) and TIGRFAM (Haft et al., 2002) databases. Motif scans were performed against PROSITE (Hulo et al., 2008) and Pfam (Finn et al., 2010) databases. Genomes of C. autoethanogenum, C. ijungdahlii, and C. ragsdalei contain several genes encoding enzymes with alcohol and aldehyde dehydrogenase activity. As indicated in Tables 7 through 10, some of these genes were found to have high homology of over 70% to butyraldehyde and butanol dehydrogenases characterized by C. acetobutylicum, C. beijerínckii, or C. saccharobutylicum,5 whereas others have at least about 40% identity with these enzymes. All three genomes encode exactly one enzyme with acetyl/butyric phosphate transferase activity and one with acetate/butyrate kinase activity. C. autoethanogenum, C. Ijungdahlii and C. ragsdalei each possess 2 aldehyde oxidoreductase genes. Table 7: C. autoethanogenum Genes Potentially Conferring Butanol and Butyl Dehydrogenase Activity Table 8: C. liunadahlii Genes Potentially Conferring Butyraldehyde and Butanol Dehydrogenase Activity Table 9: C. ragsdalei Genes Potentially Conferring Butyraldehyde and Butanol Dehydrogenase Activity

Gene Expression Studies

Gene expression studies were performed to confirm the successful expression of the introduced thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, electron transfer flavoprotein A, and electron transfer flavoprotein B genes in C. autoethanogenum harboring the butanol plasmid pM-TL85245-thlA-crt-hbd. In addition, a selection of putative butyraldehyde dehydrogenase, butanol dehydrogenase, acetyl/butyryl phosphate transferase, acetate/butyrate kinase, aldehyde oxidoreductase, and redoxin genes identified in the C. autoethanogenum genome (Table 7) were also found to be expressed under standard fermentation conditions (Figure 60).

A sample was collected by centrifugation (6000 x g, 5 min, 4 °C). RNA was isolated by suspending the cell pellet in 100 μL of lysozyme solution (50,000 U lysozyme, 10% SDS, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8). After 5 min, 350 ml of lysis buffer (containing 10 μL of 2-mercaptoethanol) was added. The cell suspension was mechanically disrupted by passing it through an 18–21 gauge needle five times. RNA was then isolated using the PureLink™ RNA Mini kit (Invitrogen) and eluted in 100 μL of RNase-free water. RNA was verified via PCR and gel electrophoresis and quantified spectrophotometrically and treated with DNase I (Roche) if necessary. RNA quality and integrity were verified using a Bioanalyzer (Agilent Technologies). The reverse transcription step was performed using the Superscript III Reverse Transcriptase kit (Invitrogen). RT-PCR reactions were performed on the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad Laboratories) in a 15 μl reaction volume with 25 ng cDNA template, 67 nM of each of the primers (Table 11) and 1x iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA 94547, USA). Guanylate kinase and formate tetrahydrofolate ligase were used as housekeeping gene and non-template controls were included. The reaction conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s. A melting curve analysis was performed immediately after completion of RT-PCR (38 cycles from 58 °C to 95 °C at 1 °C/s) to detect primer dimerization or other amplification defects. mRNA for all heterologous genes could be successfully detected, showing that the genes are expressed. The signal for all genes was at a similar level. Table 10: Oligonucleotides for qRT-PCR

The invention has been described herein with reference to certain preferred embodiments to enable the reader to practice the invention without undue experimentation. However, one of ordinary skill in the art will readily recognize that many of the components and parameters may be changed or modified or replaced by known equivalents to some extent without departing from the scope of the invention. It should be noted that such modifications and equivalents are incorporated herein as if individually set forth. Titles, headings or the like are provided to enhance the reader's understanding of this document and should not be read as limiting the scope of the present invention.

The complete descriptions of all Applications, Patents and Publications cited above and below, if any, are hereby incorporated by reference. However, reference to any Applications, Patents and Publications in this specification is not and should not be taken as an acknowledgment or any other form of suggestion that they constitute valid prior art or part of the common general knowledge in any country of the world.

Throughout this specification and in the claims which follow, unless the context otherwise requires, the words "comprise", "comprising" and the like are to be understood in an inclusive sense, as opposed to an exclusive sense, that is, in the sense of "including but not limited to".

Claims (8)

1. A recombinant microorganism that produces 1-butanol, wherein the microorganism comprises exogenous thiolase, exogenous 3-hydroxybutyryl-CoA dehydrogenase, exogenous crotonase/crotonyl-CoA hydratase, exogenous butyryl-CoA dehydrogenase, and exogenous Electron Transport Flavoproteins A and B, wherein the exogenous thiolase, exogenous 3-hydroxybutyryl-CoA dehydrogenase, exogenous crotonase/crotonyl-CoA hydratase, exogenous butyryl-CoA dehydrogenase, and exogenous Electron Transport Flavoproteins A and B are introduced by transformation, and wherein the recombinant microorganism is derived from a parent microorganism selected from the group consisting of autologous Clostridium ethanogenum, Clostridium ljungdahlii, Clostridium coskatii and Clostridium ragsdalei. 2. Recombinant microorganism according to claim 1, characterized in that the microorganism further comprises one or more exogenous nucleic acids encoding one or more enzymes selected from butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. 3. Recombinant microorganism according to claim 1, characterized in that the microorganism further comprises one or more exogenous nucleic acids encoding one or more enzymes selected from phosphotransbutyrylase, butyrate kinase, ferredoxin-dependent aldehyde oxidoreductase and butanol dehydrogenase. 4. Recombinant microorganism according to claim 1, characterized in that it is deposited in the DSMZ under accession number DSM24138. 5. Method for the production of 1-butanol, characterized by the fact that it is by means of microbial fermentation of a gaseous substrate comprising CO, comprising adding to the substrate the recombinant microorganism as defined in any one of claims 1 to 4. 6. Method according to claim 5, characterized in that the yield of 1-butanol is at least 0.075 grams per liter of fermentation broth (g/l). 7. The method of claim 6, wherein the yield of 1-butanol is from 0.075 G/L to 20 G/L. 5 8. Method according to claim 5, characterized by the fact that the gaseous substrate comprises at least 20% to 100% CO by volume.